Models of hair cell mechanotransduction have existed for more than 30 years based on groundbreaking work done in lower vertebrates. The models, largely based on physiological data, have shaped the thinking about hair cell mechanotransduction for a generation. Recent advances both challenge and support these basic tenets by providing new insights into the molecular machinery involved in mechanotransduction. Technological advancements now allow us to investigate mechanotransduction at the single cilia and molecular level. This is necessary to bring new data into perspective with existing, traditional theories. During the past funding period, we made use of the technological advancements and developed new methods that will allow us to directly probe mammalian mechanotransduction with unprecedented resolution. These methods include stimulation of hair bundles at high rates, imaging of bundle motion at high rates (>250kHz), imaging of fluorophores with a swept field confocal system at higher rates (500-2000 fps) and we have expanded our ability to do electrophysiology, and live hair bundle imaging followed by immunohistochemical or field emission SEM on these same bundles. With these tools we will determine the functional significance of tonotopic variations observed in the mechanotransduction process, testing the hypothesis that activation and adaptation kinetics provide tuning to outer hair cells. We will directly probe the function of USH1 syndrome proteins localized to the upper tip-link insertion point, near the tops of stereocilia. We will address new hypotheses regarding the function and molecular underpinnings of slow adaptation. We will directly couple morphological measurements with functional outcomes to determine how inner hair cell hair bundles are coupled together. Doing this is fundamental to understanding the complexity that is the mechanotransduction process.